A modified motor-clutch model reveals that neuronal growth cones respond faster to soft substrates.

Neuronal growth cones sense a variety of cues including chemical and mechanical ones to establish functional connections during nervous system development. Substrate-cytoskeletal coupling is an established model for adhesion-mediated growth cone advance; however, the detailed molecular and biophysical mechanisms underlying the mechanosensing and mechanotransduction process remain unclear. Here, we adapted a motor-clutch model to better understand the changes in clutch and cytoskeletal dynamics, traction forces, and substrate deformation when a growth cone interacts with adhesive substrates of different stiffnesses. Model parameters were optimized using experimental data from Aplysia growth cones probed with force-calibrated glass microneedles. We included a reinforcement mechanism at both motor and clutch level. Furthermore, we added a threshold for retrograde F-actin flow that indicates when the growth cone is strongly coupled to the substrate. Our modeling results are in strong agreement with experimental data with respect to the substrate deformation and the latency time after which substrate-cytoskeletal coupling is strong enough for the growth cone to advance. Our simulations show that it takes the shortest time to achieve strong coupling when substrate stiffness was low at 4 pN/nm. Taken together, these results suggest that Aplysia growth cones respond faster and more efficiently to soft than stiff substrates.

Comparative Analysis of an Expandable Modular Plant and a Stick-Built TiO2 Plant.

Expandable modular plants (EMPs) can be the future of the chemical industry, especially in emerging markets. Using the relative position of points on alternative NPV-time curves from the period of 10-20 years, we compare the economics of an expandable modular TiO2 plant with that of a large-scale TiO2 plant constructed fully on site (stick-built) and present scenarios where the expandable modular approach is economically favorable over the stick-built approach. To simplify the assessment of economic viability between constructing an EMP and a stick-built plant, we introduce a measure known as the flexibility feasibility ratio-a certain ratio of unit installed capacity in an EMP to the nameplate capacity of a stick-built plant at which the NPVs are equal at all points on alternative NPV-time curves. We determine that a flexibility feasibility ratio of 0.25 is suitable to install an expandable modular TiO2 plant in Nigeria, relatively.

3D nanomechanical mapping of subcellular and sub-nuclear structures of living cells by multi-harmonic AFM with long-tip microcantilevers.

Recent developments such as multi-harmonic Atomic Force Microscopy (AFM) techniques have enabled fast, quantitative mapping of nanomechanical properties of living cells. Due to their high spatiotemporal resolution, these methods provide new insights into changes of mechanical properties of subcellular structures due to disease or drug response. Here, we propose three new improvements to significantly improve the resolution, identification, and mechanical property quantification of sub-cellular and sub-nuclear structures using multi-harmonic AFM on living cells. First, microcantilever tips are streamlined using long-carbon tips to minimize long-range hydrodynamic interactions with the cell surface, to enhance the spatial resolution of nanomechanical maps and minimize hydrodynamic artifacts. Second, simultaneous Spinning Disk Confocal Microscopy (SDC) with live-cell fluorescent markers enables the unambiguous correlation between observed heterogeneities in nanomechanical maps with subcellular structures. Third, computational approaches are then used to estimate the mechanical properties of sub-nuclear structures. Results are demonstrated on living NIH 3T3 fibroblasts and breast cancer MDA-MB-231 cells, where properties of nucleoli, a deep intracellular structure, were assessed. The integrated approach opens the door to study the mechanobiology of sub-cellular structures during disease or drug response.

Cantilever signature of tip detachment during contact resonance AFM.

Contact resonance atomic force microscopy, piezoresponse force microscopy, and electrochemical strain microscopy are atomic force microscopy modes in which the cantilever is held in contact with the sample at a constant average force while monitoring the cantilever motion under the influence of a small, superimposed vibrational signal. Though these modes depend on permanent contact, there is a lack of detailed analysis on how the cantilever motion evolves when this essential condition is violated. This is not an uncommon occurrence since higher operating amplitudes tend to yield better signal-to-noise ratio, so users may inadvertently reduce their experimental accuracy by inducing tip-sample detachment in an effort to improve their measurements. We shed light on this issue by deliberately pushing both our experimental equipment and numerical simulations to the point of tip-sample detachment to explore cantilever dynamics during a useful and observable threshold feature in the measured response. Numerical simulations of the analytical model allow for extended insight into cantilever dynamics such as full-length deflection and slope behavior, which can be challenging or unobtainable in a standard equipment configuration. With such tools, we are able to determine the cantilever motion during detachment and connect the qualitative and quantitative behavior to experimental features.

Discrimination of adhesion and viscoelasticity from nanoscale maps of polymer surfaces using bimodal atomic force microscopy.

The simultaneous excitation and measurement of two eigenmodes in bimodal atomic force microscopy (AFM) during sub-micron scale surface imaging augments the number of observables at each pixel of the image compared to the normal tapping mode. However, a comprehensive connection between the bimodal AFM observables and the surface adhesive and viscoelastic properties of polymer samples remains elusive. To address this gap, we first propose an algorithm that systematically accommodates surface forces and linearly viscoelastic three-dimensional deformation computed via Attard's model into the bimodal AFM framework. The proposed algorithm simultaneously satisfies the amplitude reduction formulas for both resonant eigenmodes and enables the rigorous prediction and interpretation of bimodal AFM observables with a first-principles approach. We used the proposed algorithm to predict the dependence of bimodal AFM observables on local adhesion and standard linear solid (SLS) constitutive parameters as well as operating conditions. Secondly, we present an inverse method to quantitatively predict the local adhesion and SLS viscoelastic parameters from bimodal AFM data acquired on a heterogeneous sample. We demonstrate the method experimentally using bimodal AFM on polystyrene-low density polyethylene (PS-LDPE) polymer blend. This inverse method enables the quantitative discrimination of adhesion and viscoelastic properties from bimodal AFM maps of such samples and opens the door for advanced computational interaction models to be used to quantify local nanomechanical properties of adhesive, viscoelastic materials using bimodal AFM.

Defining Near-Term to Long-Term Research Opportunities to Advance Metrics, Models, and Methods for Smart and Sustainable Manufacturing.

Over the past century, research has focused on continuously improving the performance of manufacturing processes and systems-often measured in terms of cost, quality, productivity, and material and energy efficiency. With the advent of smart manufacturing technologies-better production equipment, sensing technologies, computational methods, and data analytics applied from the process to enterprise levels-the potential for sustainability performance improvement is tremendous. Sustainable manufacturing seeks the best balance of a variety of performance measures to satisfy and optimize the goals of all stakeholders. Accurate measures of performance are the foundation on which sustainability objectives can be pursued. Historically, operational and information technologies have undergone disparate development, with little convergence across the domains. To focus future research efforts in advanced manufacturing, the authors organized a one-day workshop, sponsored by the U.S. National Science Foundation, at the joint manufacturing research conferences of the American Society of Mechanical Engineers and Society of Manufacturing Engineers. Research needs were identified to help harmonize disparate manufacturing metrics, models, and methods from across conventional manufacturing, nanomanufacturing, and additive/hybrid manufacturing processes and systems. Experts from academia and government labs presented invited lightning talks to discuss their perspectives on current advanced manufacturing research challenges. Workshop participants also provided their perspectives in facilitated brainstorming breakouts and a reflection activity. The aim was to define advanced manufacturing research and educational needs for improving manufacturing process performance through improved sustainability metrics, modeling approaches, and decision support methods. In addition to these workshop outcomes, a review of the recent literature is presented, which identifies research opportunities across several advanced manufacturing domains. Recommendations for future research describe the short-, mid-, and long-term needs of the advanced manufacturing community for enabling smart and sustainable manufacturing.

Nanomechanical mapping in air or vacuum using multi-harmonic signals in tapping mode atomic force microscopy.

We present a method by which multi-harmonic signals acquired during a normal tapping mode (amplitude modulated) AFM scan of a sample in air or vacuum with standard microcantilevers can be used to map quantitatively the local mechanical properties of the sample such as elastic modulus, adhesion, and indentation. The approach is based on the observation that during the tapping mode operation in air or vacuum, the 0th and 2nd harmonic signals of microcantilever vibration are encountered under most operating conditions and can be mapped with sufficient signal to noise ratio. By measuring the amplitude and phase of the driven harmonic as well as the 0th and 2nd harmonic observables, we find analytical/semi-analytical formulas that relate these multi-harmonic observables to local mechanical properties for several classical tip-sample interaction models. Least squares estimation of the local mechanical properties is performed pixel by pixel. The method is validated through computations as well as experimental data acquired on a polymer blend made of Polystyrene and Polyolefin elastomer.

Measuring viscoelasticity of soft biological samples using atomic force microscopy.

Mechanical properties play important roles at different scales in biology. At the level of a single cell, the mechanical properties mediate mechanosensing and mechanotransduction, while at the tissue and organ levels, changes in mechanical properties are closely connected to disease and physiological processes. Over the past three decades, atomic force microscopy (AFM) has become one of the most widely used tools in the mechanical characterization of soft samples, ranging from molecules, cell organoids and cells to whole tissue. AFM methods can be used to quantify both elastic and viscoelastic properties, and significant recent developments in the latter have been enabled by the introduction of new techniques and models for data analysis. Here, we review AFM techniques developed in recent years for examining the viscoelastic properties of cells and soft gels, describe the main steps in typical data acquisition and analysis protocols, and discuss relevant viscoelastic models and how these have been used to characterize the specific features of cellular and other biological samples. We also discuss recent trends and potential directions for this field.

Anisotropy vs isotropy in living cell indentation with AFM.

The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models - transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) - to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells.

Evaluation of the elastic Young's modulus and cytotoxicity variations in fibroblasts exposed to carbon-based nanomaterials.

BACKGROUND: The conventional approaches to assess the potential cytotoxic effects of nanomaterials (NMs) mainly rely on in vitro biochemical assays. These assays are strongly dependent on the properties of the nanomaterials, for example; specific surface area (SSA), size, surface defects, and surface charge, and the host response. The NMs properties can also interfere with the reagents of the biochemical and optical assays leading to skewed interpretations and ambiguous results related to the NMs toxicity. Here, we proposed a structured approach for cytotoxicity assessment complemented with cells' mechanical responses represented as the variations of elastic Young's modulus in conjunction with conventional biochemical tests. Monitoring the mechanical properties responses at various times allowed understanding the effects of NMs to the filamentous actin cytoskeleton. The elastic Young's modulus was estimated from the force volume maps using an atomic force microscope (AFM). RESULTS: Our results show a significant decrease on Young's modulus, ~ 20%, in cells exposed to low concentrations of graphene flakes (GF), ~ 10% decrease for cells exposed to low concentrations of multiwalled carbon nanotubes (MWCNTs) than the control cells. These considerable changes were directly correlated to the disruption of the cytoskeleton actin fibers. The length of the actin fibers in cells exposed to GF was 50% shorter than the fibers of the cells exposed to MWCNT. Applying both conventional biochemical approach and cells mechanics, we were able to detect differences in the actin networks induced by MWCNT inside the cells and GF outside the cell's membrane. These results contrast with the conventional live/dead assay where we obtained viabilities greater than 80% after 24 h; while the elasticity dramatically decreased suggesting a fast-metabolic stress generation. CONCLUSIONS: We confirmed the production of radical oxygen species (ROS) on cells exposed to CBNs, which is related to the disruption of the cytoskeleton. Altogether, the changes in mechanical properties and the length of F-actin fibers confirmed that disruption of the F-actin cytoskeleton is a major consequence of cellular toxicity. We evidenced the importance of not just nanomaterials properties but also the effect of the location to assess the cytotoxic effects of nanomaterials.

Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy.

The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer-range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.

High resolution subsurface imaging using resonance-enhanced detection in 2nd-harmonic KPFM.

Second harmonic Kelvin probe force microscopy is a robust mechanism for subsurface imaging at the nanoscale. Here we exploit resonance-enhanced detection as a way to boost the subsurface contrast with higher force sensitivity using lower bias voltages, in comparison to the traditional off-resonance case. In this mode, the second harmonic signal of the electrostatic force is acquired at one of the eigenmode frequencies of the microcantilever. As a result, high resolution subsurface images are obtained in a variety of nanocomposites. To further understand the subsurface imaging detection upon electrostatic forces, we use a finite element model that approximates the geometry of the probe and sample. This allows the investigation of the contrast mechanism, the depth sensitivity and lateral resolution depending on tip-sample properties.

An active balance board system with real-time control of stiffness and time-delay to assess mechanisms of postural stability.

Increased time-delay in the neuromuscular system caused by neurological disorders, concussions, or advancing age is an important factor contributing to balance loss (Chagdes et al., 2013, 2016a,b). We present the design and fabrication of an active balance board system that allows for a systematic study of stiffness and time-delay induced instabilities in standing posture. Although current commercial balance boards allow for variable stiffness, they do not allow for manipulation of time-delay. Having two controllable parameters can more accurately determine the cause of balance deficiencies, and allows us to induce instabilities even in healthy populations. An inverted pendulum model of human posture on such an active balance board predicts that reduced board rotational stiffness destabilizes upright posture through board tipping, and limit cycle oscillations about the upright position emerge as feedback time-delay is increased. We validate these two mechanisms of instability on the designed balance board, showing that rotational stiffness and board time-delay induced the predicted postural instabilities in healthy, young adults. Although current commercial balance boards utilize control of rotational stiffness, real-time control of both stiffness and time-delay on an active balance board is a novel and innovative manipulation to reveal balance deficiencies and potentially improve individualized balance training by targeting multiple dimensions contributing to standing balance.

Measuring nanoscale viscoelastic parameters of cells directly from AFM force-displacement curves.

Force-displacement (F-Z) curves are the most commonly used Atomic Force Microscopy (AFM) mode to measure the local, nanoscale elastic properties of soft materials like living cells. Yet a theoretical framework has been lacking that allows the post-processing of F-Z data to extract their viscoelastic constitutive parameters. Here, we propose a new method to extract nanoscale viscoelastic properties of soft samples like living cells and hydrogels directly from conventional AFM F-Z experiments, thereby creating a common platform for the analysis of cell elastic and viscoelastic properties with arbitrary linear constitutive relations. The method based on the elastic-viscoelastic correspondence principle was validated using finite element (FE) simulations and by comparison with the existed AFM techniques on living cells and hydrogels. The method also allows a discrimination of which viscoelastic relaxation model, for example, standard linear solid (SLS) or power-law rheology (PLR), best suits the experimental data. The method was used to extract the viscoelastic properties of benign and cancerous cell lines (NIH 3T3 fibroblasts, NMuMG epithelial, MDA-MB-231 and MCF-7 breast cancer cells). Finally, we studied the changes in viscoelastic properties related to tumorigenesis including TGF-ß induced epithelial-to-mesenchymal transition on NMuMG cells and Syk expression induced phenotype changes in MDA-MB-231 cells.

Enhancing the optical lever sensitivity of microcantilevers for dynamic atomic force microscopy via integrated low frequency paddles.

A method is presented to enhance the optical lever sensitivity in dynamic atomic force microscopy (AFM) by nearly an order of magnitude over a wide frequency bandwidth. This is achieved by fabricating or releasing a paddle with a soft hinge close to the free end of the AFM microcantilever such that the paddle resonance frequency is well below the fundamental resonance frequency of the microcantilever. We show a significant increase in signal to noise ratio when cantilever motion is observed at the paddle for AFM systems that are not limited by thermal noise. Also, any effects due to the excitation of the second eigenmode were decoupled by locating the paddle at the node of the second eigenmode. We use these probes for higher harmonic imaging in amplitude modulated AFM (AM-AFM) on a standard polymer blend made of polystyrene and low density polyethylene. We demonstrate significantly improved contrast in higher harmonic images when observing cantilever motion at the paddle. Thus this microcantilever design can improve significantly conventional cantilever performance for dynamic AFM and is compatible with low-cost, high yield microfabrication processes.

Limit cycle oscillations in standing human posture.

Limit cycle oscillations (LCOs) are a hallmark of dynamic instability in time-delayed and nonlinear systems such as climate change models, biological oscillators, and robotics. Here we study the links between the human neuromuscular system and LCOs in standing posture. First, we demonstrate through a simple mathematical model that the observation of LCOs in posture is indicative of excessive neuromuscular time-delay. To test this hypothesis we study LCOs in the postural sway of individuals with multiple sclerosis and concussed athletes representing two different populations with chronically and acutely increased neuromuscular time-delays. Using a wavelet analysis method we demonstrate that 67% of individuals with multiple sclerosis and 44% of individuals with concussion exhibit intermittent LCOs; 8% of MS-controls, 0% of older adults, and 0% of concussion-controls displayed LCOs. Thus, LCOs are not only key to understanding postural instability but also may have important applications for the detection of neuromuscular deficiencies.

Nanoscale Mapping of Dielectric Properties of Nanomaterials from Kilohertz to Megahertz Using Ultrasmall Cantilevers.

Electrostatic force microscopy (EFM) is often used for nanoscale dielectric spectroscopy, the measurement of local dielectric properties of materials as a function of frequency. However, the frequency range of atomic force microscopy (AFM)-based dielectric spectroscopy has been limited to a few kilohertz by the resonance frequency and noise of soft microcantilevers used for this purpose. Here, we boost the frequency range of local dielectric spectroscopy by 3 orders of magnitude from a few kilohertz to a few megahertz by developing a technique that exploits the high resonance frequency and low thermal noise of ultrasmall cantilevers (USCs). We map the frequency response of the real and imaginary components of the capacitance gradient (∂C(ω)/∂z) by using second-harmonic EFM and a theoretical model, which relates cantilever dynamics to the complex dielectric constant. We demonstrate the method by mapping the nanoscale dielectric spectrum of polymer-based materials for organic electronic devices. Beyond offering a powerful extension to AFM-based dielectric spectroscopy, the approach also allows the identification of electrostatic excitation frequencies which affords high dielectric contrast on nanomaterials.

The relationship between intermittent limit cycles and postural instability associated with Parkinson's disease.

BACKGROUND: Many disease-specific factors such as muscular weakness, increased muscle stiffness, varying postural strategies, and changes in postural reflexes have been shown to lead to postural instability and fall risk in people with Parkinson's disease (PD). Recently, analytical techniques, inspired by the dynamical systems perspective on movement control and coordination, have been used to examine the mechanisms underlying the dynamics of postural declines and the emergence of postural instabilities in people with PD. METHODS: A wavelet-based technique was used to identify limit cycle oscillations (LCOs) in the anterior-posterior (AP) postural sway of people with mild PD (n = 10) compared to age-matched controls (n = 10). Participants stood on a foam and on a rigid surface while completing a dual task (speaking). RESULTS: There was no significant difference in the root mean square of center of pressure between groups. Three out of 10 participants with PD demonstrated LCOs on the foam surface, while none in the control group demonstrated LCOs. An inverted pendulum model of bipedal stance was used to demonstrate that LCOs occur due to disease-specific changes associated with PD: time-delay and neuromuscular feedback gain. CONCLUSION: Overall, the LCO analysis and mathematical model appear to capture the subtle postural instabilities associated with mild PD. In addition, these findings provide insights into the mechanisms that lead to the emergence of unstable posture in patients with PD.

Substrate Deformation Predicts Neuronal Growth Cone Advance.

Although pulling forces have been observed in axonal growth for several decades, their underlying mechanisms, absolute magnitudes, and exact roles are not well understood. In this study, using two different experimental approaches, we quantified retrograde traction force in Aplysia californica neuronal growth cones as they develop over time in response to a new adhesion substrate. In the first approach, we developed a novel method, to our knowledge, for measuring traction forces using an atomic force microscope (AFM) with a cantilever that was modified with an Aplysia cell adhesion molecule (apCAM)-coated microbead. In the second approach, we used force-calibrated glass microneedles coated with apCAM ligands to guide growth cone advance. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. Both approaches showed that Aplysia growth cones can develop traction forces in the 10(0)-10(2) nN range during adhesion-mediated advance. Moreover, our results suggest that the level of traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism previously observed in other cell types. Interestingly, the absolute level of traction force did not correlate with growth cone advance toward the adhesion site, but the amount of microneedle deflection did. In cases of adhesion-mediated growth cone advance, the mean needle deflection was 1.05 ± 0.07 µm. By contrast, the mean deflection was significantly lower (0.48 ± 0.06 µm) when the growth cones did not advance. Our data support a hypothesis that adhesion complexes, which can undergo micron-scale elastic deformation, regulate the coupling between the retrogradely flowing actin cytoskeleton and apCAM substrates, stimulating growth cone advance if sufficiently abundant.